Carbohydrate components of woody biomass (cellulose and hemicellulose) represent an abundant potential source of sugars for microbial conversion into renewable fuels, plastics, and other chemicals (Carole et al., 2004; Jarboe et al., 2007; Jarboe et al., 2010; Saha et al., 2003). However, cost-effective depolymerization of this complex material to produce fermentable sugar streams remains a major challenge (Alvira et al., 2010; Saha et al., 2003). Pretreatment processes such as diluting mineral acids at elevated temperature and pressures open the structure of woody biomass to increase the effectiveness of cellulase enzymes, and hydrolyze the pentose polymers of hemicellulose into monomers. Unwanted side reactions from this pretreatment also produce a mixture of compounds (furans, acetate, soluble products from lignin, and others) that inhibit growth and retard fermentation (Almeida et al., 2009; Jarboe et al., 2007; Mills et al., 2009). Most inhibitors can be removed or neutralized by separating the solubilized sugars from the cellulose-enriched fiber using counter-current washing followed by over-liming (Martinez et al., 2001; Martinez et al., 2000a). However, these added process steps would also add cost to renewable products. By developing robust biocatalysts that are resistant to side products from pretreatment it should be possible to design a simpler process (Geddes et al., 2010a, b).
Furfural, the dehydration product of xylose, is of particular importance as a fermentation inhibitor in hemicellulose hydrolysates (Almeida et al., 2009; Mills et al., 2009). Furfural concentrations in hemicellulose hydrolysates have been correlated with toxicity (Zaldivar et al., 1999). The addition of furfural to over-limed hemicellulose hydrolysates has been shown to restore toxicity (Martinez et al., 2001; Martinez et al., 2000a). In model studies with various hydrolysate inhibitors, furfural was unique in potentiating the toxicity of other compounds (Zaldivar et al., 1999). Furan alcohols (reduced products) are less toxic than the respective aldehydes (Zaldivar et al., 2000; Zaldivar et al., 1999). Furfural-resistant mutants of ethanologenic Escherichia coli have been isolated and characterized (Miller et al., 2009a, b; Turner et al., 2010). Resistance to low concentrations of furfural was found to result from the silencing of yqhD, an NADPH-dependent, furfural oxidoreductase that is induced by furfural (Miller et al., 2009a, b; Turner et al., 2010). Although there are multiple NADPH-furfural reductases in E. coli and conversion of furfural to the less toxic alcohol which would be generally regarded as beneficial, the unusually low Km of YqhD for NADPH appears to create a metabolic conflict by competing with biosynthesis for NADPH (Miller et al., 2009a). Metabolic routes for the anaerobic production of NADPH during xylose fermentation are quite limited (Frick et al., 2005; Grabowska et al., 2003; Milles et al., 2009). The metabolism of furfural by YqhD is proposed to inhibit growth and fermentation by depleting the pool of NADPH below that required for essential biosynthetic reactions (Miller et al., 2009a, b; Turner et al., 2010). Sulfate assimilation was identified as a site that is particularly sensitive to NADPH limitation (Miller et al., 2009a). Furan toxicity (furfural and 5-HMF) can be minimized by a variety of approaches that increase the availability of NADPH (Miller et al., 2009a, b; Miller et al., 2010). The pntAB-encoded, membrane transhydrogenase has also been shown to increase furfural tolerance by directly supplying NADPH using NADH as the electron donor (Miller et al., 2009a, b; Miller et al., 2010).
Low levels of NADH-dependent oxidoreductases appear to be present in crude extracts of E. coli. Furfural reduction by these should not affect the NADPH and would eliminate competition during biosynthesis. One gene, fucO, has been previously discovered to be an NADH-dependent oxidoreductase that reduces furfural and hydroxymethyl furfural. Increased expression of fucO was shown to increase cell tolerance to furfural (Wang et al., 2011a).
Further studies were undertaken to identify additional NAD(P)H-oxidoreductases that could reduce furfural and confer increased furfural tolerance using expression arrays that examine the entire genome. These were not successful. Instead, we made the unexpected discovery of a novel gene (ucpA) that confers furfural tolerance by an unknown mechanism, although UcpA has a putative NAD(P)H-binding site and shares some similarity with some short chain oxidoreductases (Sirko et al., 1997) that allowed identification as a candidate gene. There is very little published literature about UcpA. UcpA does not encode furfural reductase activity based on in vitro assays and whole cell (in vivo) assays. UcpA also does not exhibit transhydrogenase activity. The mechanism of UcpA action that leads to increased furfural tolerance remains unknown.
Reduction of educe furfural with NADH or increased production of NADPH have been shown to be effective approaches to increase furfural tolerance (Miller et al., 2009a, b; Miller et al., 2010; Wang et al., 2011a) and a need for providing various means for increasing furfural resistance in genetically modified microorganisms remains.